Techniques for studying Metal-Drug- DNA interactions

Study of the interaction between the metal complexes

Techniques used to Study MetalloDrugsDNA interactions

with DNA by different techniques : Covalent Binding No Covalent : Spectrophotometric Titration by UV-vis

Maribel Navarro mnavarro@ivic.gob.ve IVIC-Venezuela Brasil-Florianpolis 26-30 Julio 2010

Metal Complexes against Different Diseases

DNA : important target for metal complexes

Leishmania
COVALENT

This kind of interaction involves covalent bond formation of the metal complex to either the phosphate or the nucleic acid bases

Trypanosomiasis

Cancer

Malaria
Mini-Reviews in Medicinal Chemistry, 2004, 4, 23-30

Covalent binding of the cisplatin to the ADN

Covalent inner-sphere binding

This kind of interaction involves covalent bond

formation of the metal complex to either the phosphate or the nucleic acid bases.
Where more than one metal - DNA bond is
1,2-Intrastrand 1,2-Interstrand

formed, crosslink results which can be either intra- or inter-strand.

ADN

1,2-Intrastrand

1,2-Interstrand
Coor. Chem. Rev. 216-217, 383,( 2001)

Covalent binding of the cisplatin to the ADN

Covalent binding of the metal complexes to the ADN

In the Laboratory

Covalent binding
Precipitation EtOH, NaCl Then redisolution

Incubation 120 h

Metal Complex

DNA solution Metal Quantification (AA o ICP)

nmol Metal/mg ADN metal

DNA Quantification (UV-Vis)

Burton Assay
Quantification of [Metal]
HClO4 NH(C6H5)2 Calibration 600 nm HClO4 NH(C6H5)2 [ADN] quantification
K. Burton, Biochem. J. ,62, 315 (1956)

Metal standards

HCl, 70C

Metal samples

Covalent binding

Techniques for studying Metal-Drug- DNA interactions

Study of the interaction between the metal complexes

with DNA by different techniques : Covalent Interaction Spectrophotometric Titration by UV-vis

Complejo (1) Pt(CQDF)2(I)2, (2) Pt(CQDF)2(Cl)2 (3) Pt(CQ)2(Cl)2 (4) Pd(CQDF)2(I)2 (5) Pd(CQ)2(Cl)2 (6) Au(CQ)(Cl) (7) Au(CQ)(TgTa) (8) [Au(CQDF)(PPh3)]PF6 cis-Pt(NH3)2(Cl)2 trans-Pt(NH3)2(Cl)2

nmol metal/mg ADNtt 183,41 171,72 721,49 125,07 1359,21 1420,75 531,52 10,91 1136,52 1135,47

Base Pair /metal (P.B./A.M.) 8,39 8,96 2,13 12,32 1,13 1,03 2,89 140,98 1,45 1,46

DNA : important target for metal complexes

Non-covalent outer sphere binding

Negatively charged backbone of DNA interacts
Hydrogen bonding

NO COVALENT

Electrostatic interactions Minor or Major Groove Intercalation.

with positively charged molecules through electrostatic interactions or phosphate - oxygen binding. Exocyclic groups on the purines/pirimidines can be involved trough hydrogen bonding to suitable ligand atoms. Depends primary, on the nature and concentration of the metal. measured by Tm and CD.

No covalent interaction

Mutat Res. 623, 3, (2007)

Interaccin no covalente

Pt

OH

Mutat Res. 623, 3, (2007)

Electronic Excitation by UV/Vis Spectroscopy :

An Electronic Spectrum
1.0 maxwith certain extinction UV
Make solution of concentration low enough that A 1

Visible

(Ensures Linear Beers law behavior) Even though a dual beam goes through a solvent blank, choose solvents that are UV transparent.

Absorbance

Can extract the value if conc. (M) and b (cm) are known UV bands are much broader than the photonic transition event. This is because vibration levels are superimposed on UV.

Hypsochromic shift

Bathochromic shift

0.0 200

400 Wavelength, , generally in nanometers (nm)

800

UV-vis
* and * transitions: high-energy, accessible in vacuum UV (max <150 nm). Not usually observed in molecular UV-Vis. n * and * transitions: non-bonding electrons (lone pairs), wavelength (max) in the 150-250 nm region. n * and * transitions: most common transitions observed in organic molecular UV-Vis, observed in compounds with lone pairs and multiple bonds with max = 200-600 nm. Metal Ligand Charge Transitions (MLCT) or Metal Ligand Charge Transitions (LMCT)

CT *

CT * MLCT d*

MLCT d*

N N N N Ru
2+

N N

(PF6)2
NH2 NH2

(PF6)2

N N N N Ru
2+

N N

O O

Buffer Tris-HCl(5mM), NaCl(50mM) pH 7.2

Buffer Tris-HCl(5mM), NaCl(50mM) pH 7.2

Studies of no covalent interaction

0.6

Grfica absorbancia Vs. longitud de onda del complejo [Au(CQ)(PPh3)] PF6 en buffer (comportamiento durante la titulacin con ADN) 0 uL
DNA 5 uL DNA 10 uL DNA

Spectroscopic Titrations
DNA Aliquots

0.5

Absorbancia

0.4

0.3

0.2

0.1 270 290 310 330 Longitud de onda (nm) 350

Metal Complex at fixed concentration

Neighbor exclusion equation.

[ADN]/(a-f) = [ADN]/(a-b) + 1/[Kb(a-b)] %H= (Ai-Af/Ai)100

[DNA]/( a- f) = [DNA]/(a- b) + 1/[Kb(a-b)] The intrinsic binding constant Kb was determined from the plot of [DNA]/(ea - ef) vs [DNA], where [DNA] is the concentration of DNA in base pairs the apparent absorption coefficients, a, f, b correspond to Aobs/[M], the extinction coefficient for the free metal complex and the extinction coefficient of the metal complex in the totally bound form. The slope equal to 1/(b - f) and the intercept equal to 1/[Kb(b - f)] and Kb was obtained adjusting the data to the corresponding curve

[ADN]/(a-f) = [ADN]/(a-b) + 1/[Kb(a-b)]

[ADN]/(a-f) = [ADN]/(a-b) + 1/[Kb(a-b)]

3.5E-08

3.0E-08

Kb = Slope/ Cut off

y = 0,0009x + 2E-09 R2 = 0,999
Slope Cut off point 9.00E-04 2.00E-09 4.50E+05 7.48
3.5E-05 4.0E-05

2.5E-08

2.0E-08

1.5E-08

[ADN] / (ea-ef)

Kb %hip
1.0E-08 1.0E-05 1.5E-05 2.0E-05

[ADN] 2.5E-05

3.0E-05

Spectroscopic titration
Scatchards Equation

DNA

Complex metal
Scatchards Equation

0.5

Grfica absorbancia Vs. longitud de onda del complejo [Au(CQ)(PPh3)] PF6 en buffer (comportamiento durante la titulacin con 0 uL ADN)
D NA

Absorbancia

0.3

0.1 270 290 310 330 Longitud de onda (nm) 350

Data from UV-vis experiment

Compounds
2.5E+06 2.3E+06 2.1E+06 1.9E+06 1.7E+06

CQDP [Au(CQ)(PPh3)] PF6

y = -3.02E+07x + 7.48E+06 R = 0.897

Kb1(x107 M-1) 1.38 0.56 3.79 0.01

Kb1(x105 M-1) 1.02 0.15 1.84 0.89

Constant binding
Complex
% Hypochromism Bathochromi sm (nm)

Neighbor exclusion
5 -1 Kb(x10 M )

Scatchard
5 -1 Kb2(x10 M ) 7 -1 Kb1(x10 M )

Au(CQ)(Cl)
[Au(CQ)(PPh3)]PF6

21% 7.5% 31% 24%

4 3 5 2

2,68 0,09 4.50 0.25 0,79 0.62 1,02 0,15

2.37 0.76 3.79 0.01 0.51 0.25 0.93 0.21

4.03 0.87 1.84 0.25 0.92 0.61 1.38 0.55

v/[L]

1.5E+06 1.3E+06 1.1E+06 9.0E+05 7.0E+05 5.0E+05 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4

[Au(CQDF)(PPh3)]PF6
y = -2.01E+05x + 7.08E+04 R = 0.71

CQDF
v

Thermal denaturation of DNA

Metal Complex/DNA Ri= 0.2

Number

Start 40.0 oC 55.0 oC 70.0 oC

Stop 55.0 oC 70.0 oC 94.0 oC

Spectrofotometric Titration of [Au(CQ)(PPh3)]PF6 with DNA. [Complejo] = 9,98x10-4 M and [DNA] = 050 M

Fluorimetrc Titration [Au(CQ)(PPh3)]PF6 with DNA. [Complejo] = 7,32x10-4 M and [DNA] = 010 M

1 2 3

Incremen t 3.0 oC 1.0 oC 3.0 oC

Hold time 0.5 min 0.5 min 0.5 min

Compounds CQDP [Au(CQ)(PPh3)] PF6

UV-Vis Titration Kb1(x107 M-1) Kb1(x105 M-1) 1.38 0.56 1.02 0,15 3.79 0.01 1.84 0.89

Fluorescence Titration Kb1(x107 M-1) Kb2(x105 M-1) 3.24 1.21 3.26 1.01 5.27 1.82 2.44 1.69

Tm

Tm

DNA Melting Temperature after interaction with metal complex

Complex
Au(CQ)(Cl)

Tm
69,6 0,6 80,6 0,4 85,4 1,5 64,9 0,1 87,6 0.9

Tm
4,7 15,7 20,5 --22,7

[Au(CQ)(PPh3)] PF6
[Au(CQDF)(PPh3)]PF6 DNA CQDP